Over-expression, purification, and characterization of protein pGEM-gbr22 in non-lethal E. coli bacteria
In order for a foreign protein to be expressed in Escherichia coli, used frequently due to its availability and low price, a plasmid containing the protein must transform the cell culture to be resistant against ampicillin[1]. The ampicillin-resistant protein allows all E. coli cells not containing the purple gbr22 to die off for better analysis of the target protein. Lysozyme was used to break down cell walls to allow easy transfer of the plasmid, albeit after heat-shocking [2]. Purification through chromatography is effective at isolating the target protein through use of histidine tags. Nanodrop spectroscopy and gel electrophoresis (SDS-PAGE) are used to characterize the protein. This lengthy process is used to determine the purity and molecular weight of the protein, gbr22, while isolating it from all other proteins that could create a false molecular weight or contaminate the protein wanted for isolation.
A tube containing 25 μL of competent bacterial cells (non-lethal E. coli BL21 (DE3), New England Biolabs, Ipswich, Massachusetts, United States) and plasmid were heat-shocked in a water bath at 42 °C for 45 s, iced, and SOC media was added. The cells were placed on a plate and left to incubate overnight at 37 °C. Ten microlitres of ampicillin and 5 mL of LB broth were added to the tube and left to shake and incubate for roughly 8 h at 300 rpm and 37 °C. Ampicillin was later added. Part of that was saved as sample one; the rest was centrifuged and later resuspended in PBS and lysozyme, and placed in the -20 °C freezer. Once thawed, benzonase was added, centrifuged, and the supernatant liquid was drawn off. The supernatant liquid was kept as sample two. The sample was run through a filter column, washed, and eluted twice, collected as different samples. A nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, United States) was then used to analyze the absorbance of the two elutions at 280 nm and 574 nm, from a very small 2 µL sample. Remaining samples were centrifuged once more, and loading buffer was added, mixing the samples well. The samples were then heated at 95 °C for a few minutes and then centrifuged again. These samples were used in comparison against a molecular weight standard used in SDS-PAGE wells, run for 25 min at 200 V. Once the gel was run, it was washed repeatedly, stained, and dried. The gel did not survive the drying process.
Results:
Figure 1. E. coli BL21 (DE3) cells with no DNA control, after 24 h of incubation at 37 °C. Very few colonies exist on the plate because of the effect the antibiotic ampicillin has on the cells, namely, it does not allow them to grow.
Figure 2. E. coli BL21 (DE3) colonies after 24 h of incubation at 37 °C. Cell colonies were propagated because the protein expressed by the bacteria, pGEM-gbr22, is ampicillin-resistant.
Figure 3. Incubation of cells, after 24 h at 37 °C, from inside the human mouth failed on this "fun plate" because of human error in choosing this plate, treated with ampicillin, when a plate without ampicillin should have been chosen.
Figure 4. Erlenmeyer flask of pGEM-gbr22 protein in a solution of ampicillin and LB broth. The protein was expressed, hence the purple color, after 24 h of incubation at 37 °C in a shaking water bath.
Figure 5. E. coli expressing pGEM-gbr22 protein after centrifuging of the sample and removal of the resulting supernatant liquid.
Figure 6. 15 mL conical tubes containing elution one (left), which comprises most of the expressed protein, after being run through a Ni-NTA column; elution two (right) contains that which remains after elution one is collected, and the Ni-NTA column is washed through again.
Figure 7. Data from the nanodrop spectrophotometer, analyzing elution one at a wavelength of 280 nm. Using Beer's Law (A = εbc ∴ c = A/εb), the concentration of the protein is 0.801/38 850 = 2.06E-5 M.
Figure 8. Data from the nanodrop spectrophotometer, analyzing elution one at a wavelength of 280 nm. Using Beer's Law (A = εbc ∴ c = A/εb), the concentration of the protein is 0.780/38 850 = 2.00E-5 M.
Figure 9. Data from the nanodrop spectrophotometer, analyzing elution one at a wavelength of 574 nm. The 10 mm path resulted in an absorbance level of 0.159 mg/ml. (n=4)
Figure 10. Data from the nanodrop spectrophotometer, analyzing elution one at a wavelength of 574 nm. The 10 mm path resulted in an absorbance level of 0.126 mg/ml. (n=4)
Figure 11. Data from the nanodrop spectrophotometer, analyzing elution one at a wavelength of 574 nm. The 10 mm path resulted in an absorbance level of 0.125 mg/ml. (n=4)
Figure 12. Data from the nanodrop spectrophotometer, analyzing elution one at a wavelength of 574 nm. The 10 mm path resulted in an absorbance level of 0.124 mg/ml. (n=4)
Figure 13. Sample ladder that would result from SDS-PAGE, to be used as a comparison to determine the molecular weight of proteins. This particular ladder can determine proteins between a minimum weight of 10 kDa and a maximum weight of 170 kDa.
Figure 14. Gel with ladder in well one for comparison, then wells two through seven contain samples one through six of experimenter one; wells eight through ten contain samples four through six of experimenter two. Purple bands (due to staining) represent extra proteins settled in at their respective molecular weights. The darkest band is the main pGEM-gbr22 protein.
Error could have stemmed from any one of the many steps in this lab. Lysozyme may have been added twice, doubling its concentration, and potentially affecting the E. coli's uptake of the plasmid, and, eventually, the expression of the purple gbr22 protein. Pipets used during the spectrophotometry phase may have been contaminated with the slightest touch of a foreign object, rendering all data based on the nanodrop useless and inaccurate. The samples run through the Ni-NTA column may have had foreign protein in them, ruining the molecular weight estimate and purity estimate. The purity of the protein, with included errors, is believed to be around 50%.
Sample one contained whole E. coli cells from the initial protein expression and harvest, often centrifuged down to a pellet of organic matter. The lysate (collected after the cells were lysed) comprised sample two. Sample three was waste washed through the Ni-NTA column. After being run through the Ni-NTA column and attracted to the histidines (via imidazole), the resulting protein liquid, after being washed, made up sample four. Sample five was the protein liquid (sample four) eluted (protein was washed) once; sample six was sample four eluted twice. The elution buffer had more imidazole (and, thus, histidines) than the wash buffer, better separating the protein from the waste.
During these three labs, over the course of a few weeks, the protein pGEM-gbr22—found in coral off the Great Barrier Reef in Australia—was expressed, purified, and characterized. The molecular weight and concentration of the protein could be determined at various steps in these labs, and was ultimately estimated to be about 25 kDa. Future labs could use these results and products in enzyme assays to better understand the nature of the gbr22 protein.
References:
Protein production and purification. Nat Methods.2008, 5(2), 135-146.
Over-expression, purification, and characterization of protein pGEM-gbr22 in non-lethal E. coli bacteria
In order for a foreign protein to be expressed in Escherichia coli, used frequently due to its availability and low price, a plasmid containing the protein must transform the cell culture to be resistant against ampicillin[1]. The ampicillin-resistant protein allows all E. coli cells not containing the purple gbr22 to die off for better analysis of the target protein. Lysozyme was used to break down cell walls to allow easy transfer of the plasmid, albeit after heat-shocking [2]. Purification through chromatography is effective at isolating the target protein through use of histidine tags. Nanodrop spectroscopy and gel electrophoresis (SDS-PAGE) are used to characterize the protein. This lengthy process is used to determine the purity and molecular weight of the protein, gbr22, while isolating it from all other proteins that could create a false molecular weight or contaminate the protein wanted for isolation.
A tube containing 25 μL of competent bacterial cells (non-lethal E. coli BL21 (DE3), New England Biolabs, Ipswich, Massachusetts, United States) and plasmid were heat-shocked in a water bath at 42 °C for 45 s, iced, and SOC media was added. The cells were placed on a plate and left to incubate overnight at 37 °C. Ten microlitres of ampicillin and 5 mL of LB broth were added to the tube and left to shake and incubate for roughly 8 h at 300 rpm and 37 °C. Ampicillin was later added. Part of that was saved as sample one; the rest was centrifuged and later resuspended in PBS and lysozyme, and placed in the -20 °C freezer. Once thawed, benzonase was added, centrifuged, and the supernatant liquid was drawn off. The supernatant liquid was kept as sample two. The sample was run through a filter column, washed, and eluted twice, collected as different samples. A nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, United States) was then used to analyze the absorbance of the two elutions at 280 nm and 574 nm, from a very small 2 µL sample. Remaining samples were centrifuged once more, and loading buffer was added, mixing the samples well. The samples were then heated at 95 °C for a few minutes and then centrifuged again. These samples were used in comparison against a molecular weight standard used in SDS-PAGE wells, run for 25 min at 200 V. Once the gel was run, it was washed repeatedly, stained, and dried. The gel did not survive the drying process.
Results:
Error could have stemmed from any one of the many steps in this lab. Lysozyme may have been added twice, doubling its concentration, and potentially affecting the E. coli's uptake of the plasmid, and, eventually, the expression of the purple gbr22 protein. Pipets used during the spectrophotometry phase may have been contaminated with the slightest touch of a foreign object, rendering all data based on the nanodrop useless and inaccurate. The samples run through the Ni-NTA column may have had foreign protein in them, ruining the molecular weight estimate and purity estimate. The purity of the protein, with included errors, is believed to be around 50%.
Sample one contained whole E. coli cells from the initial protein expression and harvest, often centrifuged down to a pellet of organic matter. The lysate (collected after the cells were lysed) comprised sample two. Sample three was waste washed through the Ni-NTA column. After being run through the Ni-NTA column and attracted to the histidines (via imidazole), the resulting protein liquid, after being washed, made up sample four. Sample five was the protein liquid (sample four) eluted (protein was washed) once; sample six was sample four eluted twice. The elution buffer had more imidazole (and, thus, histidines) than the wash buffer, better separating the protein from the waste.
During these three labs, over the course of a few weeks, the protein pGEM-gbr22—found in coral off the Great Barrier Reef in Australia—was expressed, purified, and characterized. The molecular weight and concentration of the protein could be determined at various steps in these labs, and was ultimately estimated to be about 25 kDa. Future labs could use these results and products in enzyme assays to better understand the nature of the gbr22 protein.
References: